Despite the dual role for TGF-beta as both tumor suppressor and tumor promoter in carcinogenesis, preclinical data from our lab and others has previously suggested that strategies to antagonize TGF-beta may selectively reduce the undesirable tumor promoting effects of this growth factor while sparing the desirable effects on tumor suppression and normal homeostasis. Based on these promising preclinical results, several different TGF-beta pathway antagonists are in early phase clinical trials for the treatment of advanced cancer. However, given the complex biology of TGF-beta, the successful development of TGF-beta antagonists for cancer therapy will depend on a clear understanding of how these agents work, and the related question of how to select patients who will benefit from this type of treatment. Our work this year has fallen into three main areas: 1. OPTIMIZATION OF ANTI-TGF-BETA THERAPY AND DEVELOPMENT OF PREDICTIVE BIOMARKERS. One major focus in FY13 has been to work with a panel of transplantable syngeneic mouse models of metastatic breast cancer to develop predictive biomarkers of response to anti-TGF-beta antibody therapy, and to explore combination therapy with conventional chemotherapeutics in order to improve overall efficacy. Metastatic burden is our primary therapeutic endpoint. Using this tumor panel, we have found that while TGF-beta antagonism inhibits metastasis in some models, it has no effect on or can even stimulate metastasis in other models. We have excluded a number of plausible candidate predictive biomarkers of response to TGF-beta antagonism and work with discovery-based approaches is ongoing, including testing gene signatures of TGF-beta-mediated tumor suppression that we identified in the related project ZIA BC 005785. We are also approaching this problem by investigating the mechanisms that underlie the undesirable effects of TGF-beta antagonism that are seen in some models. Unlike the therapeutic effects of TGF-beta antibodies which largely involve reactivation of effective anti-tumor immune responses, we have shown that the undesirable stimulatory effects are independent of the immune system, and seem to involve neutralization of residual tumor suppressive effects of TGF-beta on the tumor parenchyma. In the area of combination therapy, we have shown that the therapeutic efficacy of TGF-beta antibodies can be enhanced by combination with subtherapeutic doses of immunomodulatory chemotherapeutics, leading to a clear survival benefit. Work with other therapeutic combinations is ongoing. As part of this project, we also uncovered a novel functional role for a B-cell marker, CD79a, in the metastasis-promoting effects of immature myeloid cells. 2. DEVELOPMENT OF TOOLS TO IDENTIFY AND QUANTITATE NON-CANONICAL TGF-BETA SIGNALING. Recent data from other labs has suggested that TGF-beta signaling via non-canonical mechanisms is associated with tumor promoting effects of TGF-beta, while canonical signaling is critical for tumor suppressor effects. Thus activation of non-canonical TGF-beta signaling may contribute to the switching of TGF-beta from tumor suppressor to tumor promoter. Non-canonical signaling mechanisms include the formation of 'mixed Smad' signaling complexes involving both TGF-beta Smads (Smad2/3) and BMP Smads (Smad1/5/8). In situ proximity ligation (Duolink) is a new technique that uses paired antibodies to detect and quantitate protein/protein interactions in frozen or fixed tissue sections. We have generated and validated antibodies for the detection of the mixed Smad complexes, and have successfully developed the first bright-field application of this technique for application to formalin-fixed, paraffin-embedded tissue and thus to archived clinical specimens. We have show that mixed Smad complexes are highly expressed in the embryo but not normal adult tissues, and are reactivated in disease states. We are applying this approach to look at the onset of non-canonical signaling at different stages in cancer progression in pre-clinical and clinical breast cancer material. We are also screening our metastatic breast cancer panel to determine whether non-canonical TGF-beta signaling might serve as a useful predictive biomarker for therapy with TGF-beta antagonists. 3. TGF-BETA ISOFORM EFFECTS IN TUMORIGENESIS. There are three isoforms of TGF-beta with very similar activities in vitro. Correlative clinical data and literature evidence suggests that whereas TGF-beta1 is primarily associated with poor outcome, TGF-beta3 may actually oppose TGF-beta1 and be associated with good outcome. To address this question, we are collaborating with XOMA Corp to assess the relative efficacy in our metastasis models of therapeutic anti-TGF-beta antibodies with different isoform selectivity. In parallel, we are performing detailed characterization of the TGF-beta isoform expression profiles of the different models. Through this approach, we hope to generate improved TGF-beta-targeted therapeutics.